QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online November 23, 2005
doi: 10.1242/10.1242/jcs.02643

This Article Summary Full Text Full Text (PDF) An erratum has been published Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, T.-A. Articles by Avraham, S. Search for Related Content PubMed PubMed Citation Articles by Kim, T.-A. Articles by Avraham, S.

The BTB domain of the nuclear matrix protein NRP/B is required for neurite outgrowth

Tae-Aug Kim^*, Shuxian Jiang^*, Seyha Seng, Kiweon Cha, Hava Karsenty Avraham and Shalom Avraham

Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, MA 02115, USA

View larger version (27K): [in a new window]   Fig. 1. Three-dimensional modeling and site-directed mutagenesis of NRP/B. (A) Schematic presentation of primary sequence analysis of NRP/B. IVS, intervening sequence. (B) Three-dimensional modeling and mutation in the BTB domain. BTB mutant A includes mutation of Asp(D)47 to Ala(A). BTB mutant A also has mutations in specific conserved residues His(H)60 to Ala(A) and Arg(R)61 to Asp(D). Red color indicates -helix and yellow color indicates the ß-sheet structure. Mutation sites are shown as white balls. (C) Equilibrium binding analysis of NRP/B. Purified NRP/B-(His)6 bound to a HisSorb 96 plate and total cell lysate obtained from mutant NRP/B-transfected cells were incubated in the same plate. The dimerized NRP/B was detected by anti-GFP antibody (upper panel). Equal amounts of NRP/B protein bound to the HisSorb 96-well plate were probed by monoclonal anti-NRP/B antibody, VD2. (D) Dimer formation between the BTB domains of NRP/B as revealed by protein crosslinking. GST sequence was proteolytically removed from recombinant NRP/B-GST, and then recombinant NRP/B was incubated in the presence (+) (lanes 1 and 2) or absence (-) (lane 3) of DSP. After quenching the residual DSP with Tris-HCl, pH 7.4, samples were run on reducing 8% SDS-PAGE (lane 2) or non-reducing 8% SDS-PAGE (lanes 1 and 3), and then visualized by staining with Coomassie Blue. Arrows indicate monomeric (mono) and dimeric (di) NRP/B. BTB mtA, BTB mutant A. Positions of molecular size markers are indicated in kDa on the right.

View larger version (18K): [in a new window]   Fig. 2. The NRP/B-BTB domain is essential for nuclear localization. Transient transfection of NRP/B domains in PC12 cells. NRP/B cDNA constructs were generated in a pEGFP-N2 vector for transfection into PC12 cells. Cells were transiently transfected with pEGFP-N2 vector alone, or with pEGFP-N2-NRP/B-BTB domain or NRP/B kelch domain, as illustrated below micrographs. Transfected PC12 cells were observed by their green fluorescent color using UV microscopy. Bars, 10 µm.

View larger version (33K): [in a new window]   Fig. 3. NRP/B interaction with p110RB is mediated via the BTB domain. (A) The association of the NRP/B-BTB domain with p110RB. NRP/B-GFP constructs were cotransfected with pRBWtHA-SVE constructs into 293T cells. Total cell lysates were immunoprecipitated with anti-HA monoclonal antibody. NRP/B association with pRB was observed by probing with anti-GFP polyclonal antibody. The membrane was reprobed with anti-HA antibody (bottom panel). Lane 1, IgG control; lane 2, full-length NRP/B-GFP; lane 3, NRP/B-BTB-GFP; lane 4, NRP/B-kelch-GFP. (B) Specific association of the NRP/B-BTB domain to the B pocket of p110RB. A schematic presentation of the pRB-HA-SVE constructs (upper panel). NRP/B-GFP constructs (full-length, NRP/B-BTB, and NRP/B-kelch) were cotransfected with wild-type p110RB or with the p110RB deletion mutant (del9.1 in A pocket) into 293T cells. Total cell lysates (500 µg) in GFP buffer were immunoprecipitated with anti-HA antibody and blotted with anti-GFP monoclonal antibody or with anti-HA antibody (lower panels). pRB, p110RB.

View larger version (58K): [in a new window]   Fig. 4. Overexpression of NRP/B induced differentiation and hypophosphorylation of p110RB in Tet-on/off PC12 cells. (A) The expression of NRP/B in Tet-on/off PC12 cells. Stable NRP/B transfected PC12 Tet-on/off cells were cultured in the presence or absence of tetracycline (1 µg/ml) for 48 hours. An equal amount of protein (5x106 cells) was loaded and the blot was probed with monoclonal anti-NRP/B antibody for western blot (WB) analysis. SC10 represents the NRP/B Tet-off inducible clone and GH11 the Tet-on inducible clone. Bands indicate p67 NRP/B. (B) Morphological alteration of Tet-on PC12 cells upon tetracycline stimulation. GH11 clones were grown in 12-well plates coated with poly-D-lysine. Cells were unstimulated or stimulated with tetracycline (1 µg/ml) for 2 days and observed under a light microscope. (C) NRP/B induced hypophosphorylation of p110RB. GH11 PC12 clones were stimulated with tetracycline as above. 30 µg total cell lysates were separated by 7.5% SDS-PAGE. The blot was probed with anti-RB antibody. The slow migrated form of p110RB indicates hyperphosphorylated p110RB. Bars, 10 µm.

View larger version (30K): [in a new window]   Fig. 5. (A) Effect of NRP/B and its mutants on cell cycle progression in PC12 cells. Subconfluent cells were transfected with wild-type NRP/B and its mutants for 48 hours. Cells were then labeled with propidium iodide. Mock (pEGFP), NRP/B-GFP (wt) and mutant NRP/B-GFP transfected cells were sorted out from non-transfected cells by flow cytometric analysis. Control, non-transfected cells. The y-axis represents the percentage of the cell population at each cell cycle stage. Data are the means±s.d. of duplicate samples. (B) Inhibition of the NRP/B-p110RB association by mutation of the BTB domain. NRP/B-GFP and its mutants (BTB mutant A and kelch mutants that include mutation in the residues W518A or V4146, D418K) were cotransfected with various pRBWtHA-SVE constructs into 293T cells. The NRP/B-p110RB interaction was analyzed by immunoprecipitation with anti-HA antibody, followed by probing with anti-GFP antibody (upper panel). The membrane was reprobed with anti-HA antibody (middle panel). To determine the transfection efficiency of the NRP/B constructs, total cell lysates were immunoprecipitated with GFP antibody followed by probing with anti-GFP antibody (lower panel). BTB mtA, BTB mutant A.

View larger version (40K): [in a new window]   Fig. 6. Effect of NRP/B and its mutants on neuronal differentiation. (A) PC12 cells were grown in 10% serum as a control (left) or stimulated with NGF (right). (B) PC12 cells were transfected with either 1 µg NRP/B (WT) or NRP/B mutant, or with control GFP vector (pEGFP). Spontaneous neurite outgrowth 3 days after transfection was observed by green fluorescence using UV microscopy. The experiments were repeated at least three times. BTB mtA, BTB mutant A. (C) Quantitative analyses of neurite outgrowth in PC12 cells transfected with NRP/B and mutant NRP/B: the NRP/B-BTB domain and NRP/B-BTB mtA. The percentage of cells with neurites was calculated in representative experiments repeated three times. Data are means±s.d. Bars, 10 µm.

View larger version (54K): [in a new window]   Fig. 7. Microinjection of anti-NRP/B antibody inhibited the NGF-induced neuronal differentiation of PC12 cells. (A) PC12 cells were microinjected with purified anti-mouse IgG (panels a,b and c) or with purified anti-NRP/B antibody (panels d,e and f). pEGFP-N2 vector was co-injected as a marker. Microinjected cells were grown in the absence of NGF (panels a and d) or in the presence of NGF (50 ng/ml) (panels b,c,e and f) for 48 hours and then analyzed for morphological changes. Cells (c) and (f), in the same field as in (b) and (e), respectively, were analyzed using a fluorescent microscope. (B) Microinjection of anti-Csk antibody (as a negative control) and pEGFP-N2 into PC12 cells in the presence of NGF (50 ng/ml) for 48 hours. Microinjected cells in the same field were analyzed under a UV microscope (a) or a fluorescent microscope (b). Arrows indicate microinjected cells. (C) Quantitative analysis of neurite outgrowth upon microinjection of NRP/B antibodies. The percentage of cells with neurites either untreated or following treatment with NGF in the presence of IgG control or NRP/B antibody was calculated in representative experiments repeated three times. Data are the means±s.d. Bars, 10 µm.

View larger version (53K): [in a new window]   Fig. 8. Inhibitory effect of NRP/B siRNA on neurite outgrowth in PC12 cells. (A) PC12 cells were either untransfected (a,b) or transfected with NRP/B siRNA (c,d) or with GFP siRNA (e,f). After 48 hours, cells were untreated (-) or treated (+) with NGF (50 ng/ml) as indicated and analyzed for neurite outgrowth. (B) Quantitative analyses of neurite outgrowth by depletion of NRP/B using the NRP/B siRNA approach. NRP/B siRNA inhibited neurite outgrowth in NGF-induced PC12 cells. The percentage of cells with neurites was measured after 48 hours of NGF treatment. 300 cells were analyzed in three independent experiments for each condition. Data are the means±s.d. Significantly inhibited neurite outgrowth was observed with NRP/B siRNA and NGF compared to NGF-induced neurite outgrowth with control siRNA (*P<0.05). (C) Untransfected PC12 cells (UN), control GFP siRNA PC12 or NRP/B siRNA PC12 cells were harvested 48 hours after transfection and total mRNA was prepared. Semi-quantitative RT-PCR was performed using specific primers for NRP/B, which generated a product of 885 bp in length. As controls, we used specific primers for GAPDH and Mayven. (D) Effects of NRP/B siRNA on p110RB phosphorylation. PC12 cells were seeded on poly-D-lysine-coated plates and transfected with NRP/B siRNA or GFP siRNA as a control. After 24 hours of transfection, cells were treated with NGF (50 ng/ml) for 24 hours. Total cell lysates were prepared and 100 µg of the lysates were analyzed by 7.5% SDS-PAGE. The blots were probed with anti-RB, phospho-RB (pRB Ser795), VD2 antibodies (for NRP/B) or with CSK antibodies (an internal control), as indicated. Bars, 10 µm.